1. Field of the Invention
10-hydroxy 7-ethyl camptothecin ("HECPT") is an active metabolite of the camptothecin analog CPT-11. HECPT is also poorly soluble in water. Because of its poor water solubility, HECPT has not been directly administered by parenteral or oral routes in human subjects for the purpose of inhibiting the growth of cancer cells. This invention overcomes these limitations and claims novel pharmaceutically acceptable formulations of lactone stable HECPT, methods of administration of lactone stable HECPT, and antitumor compositions comprising solutions of lactone stable HECPT. Additionally, this invention claims novel dosages, schedules of administration, and routes of administration of HECPT formulations to humans with various forms of cancer.
2. Description of the Related Art
A. Introduction
Metabolic conversion of CPT-11 (a water soluble derivative of camptothecin) to its active metabolite 10-hydroxy 7-ethyl camptothecin (HECPT) varies from patient to patient and limits the utility of CPT-11 in achieving the highest plasma concentrations of HECPT which can be tolerated by the patient. HECPT's poor solubility in water has previously made the direct administration of HECPT impractical for the treatment of cancer. The conversion of CPT-11 to HECPT involves a putative carboxyl esterase enzyme, which is believed to be mainly responsible for the metabolic production of HECPT from CPT-11. Human lung cancer cell lines have been observed to convert less CPT-11 to HECPT than normal cells. The cancer cells' decreased metabolic conversion represents a form of resistance to CPT-11 and limits the utility of CPT-11 in terms of reliably and safely achieving adequate plasma concentrations of HECPT to inhibit the growth of cancer cells in humans.
Until now, HECPT has been considered unsuitable for direct clinical use because it is poorly soluble in water. One useful purpose of this invention is to formulate HECPT in a pharmaceutically acceptable manner using an organic solvent or a mixture of organic co-solvents to stabilize HECPT in the lactone ring form. It is this lactone stable HECPT which permits direct administration of HECPT to cancer patients. An additional purpose of this invention to provide certain indications, schedules, dosages and routes of administration of lactone stable HECPT for the purpose of treating cancer in humans.
The selection of suitable organic solvents for pharmaceutical dosage forms is limited to those which have a high degree of physiological safety. This invention describes administration of lactone stable HECPT in a pharmaceutically acceptable multi-solvent formulation, overcomes interpatient variability and drug resistance related to CPT-11 conversion to HECPT and is useful in instances where human cancer cells, because of their altered enzymatic activity, resist metabolic conversion of CPT-11 to HECPT.
B. DNA Topoisomerases
Several clinically important anticancer drugs kill tumor cells by affecting DNA topoisomerases. Topoisomerases are essential nuclear enzymes that function in DNA replication and tertiary structural modifications, such as overwinding, underwinding, and catenation, which normally arise during replication, transcription, and perhaps other DNA processes. Two major topoisomerases that are ubiquitous to all eukaryotic cells: (1) Topoisomerase I (topo I) which cleaves single stranded DNA; and (2) Topoisomerase II (topo II) which cleaves double stranded DNA. Topoisomerase I is involved in DNA replication; it relieves the torsional strain introduced ahead of the moving replication fork.
Topoisomerase I purified from human colon carcinoma cells or calf thymus has been shown to be inhibited by (a) camptothecin, (b) a water soluble analog called "CPT-11," and (c) 10-hydroxy 7-ethyl camptothecin (HECPT) which is the proposed active metabolite of CPT-11. CPT-11, camptothecin, and an additional Topo I inhibitor, topotecan, has been in used in clinical trials to treat certain types of human cancer. For the purpose of this invention, camptothecin derivatives include CPT-11, 10-hydroxy 7-ethyl camptothecin (HECPT) and topotecan. These camptothecin derivatives use the same mechanism to inhibit Topo I; they stabilize the covalent complex of enzyme and strand-cleaved DNA, which is an intermediate in the catalytic mechanism. These compounds have no binding affinity for either isolated DNA or topoisomerase I but do bind with measurable affinity to the enzyme-DNA complex. The stabilization of the topoisomerase I "cleavable complex" by camptothecin, CPT-11, or HECPT is readily reversible.
Although camptothecin, CPT-11, or HECPT have no effect on topoisomerase II, camptothecin, CPT-11 and HECPT stabilize the "cleavable complex" in a manner analogous to the way in which epipodophyllotoxin glycosides and various anthracyclines inhibit topoisomerase II.
Inhibition of topoisomerase I by camptothecin, CPT-11, or HECPT induces protein-associated-DNA single-strand breaks. Virtually all of the DNA strand breaks observed in vitro cells treated with CPT-11 or HECPT are protein linked. However, an increase in unexplained protein-free breaks can be detected in L1210 cells treated with camptothecin. The compounds appear to produce identical DNA cleavage patterns in end-labeled linear DNA. It has not been demonstrated that CPT-11, camptothecin, or HECPT cleaves DNA in the absence of the topoisomerase I enzyme.
C. Activity of HECPT, Camptothecin and CPT-11 is Cell Cycle Specific
The activity of camptothecin, CPT-11, and HECPT is cell cycle specific. The greatest quantitative biochemical effect observed in cells exposed to HECPT is DNA single-strand breaks that occur during the S-phase. Because the S-phase is a relatively short phase of the cell cycle, longer exposure to the drugs results in increased cell killing. Brief exposure of tumor cells to the drugs produces little or no cell killing, and quiescent cells are refractory. These results are likely due to two factors:
(1) The drugs inhibit topoisomerase I reversibly. Although they may produce potentially lethal modifications of the DNA structure during DNA replication, the breaks may be repaired after washout of the drug; and PA1 (2) Cells treated with topo I inhibitors, such as CPT-11 and HECPT, tend to stay in GO of the cell cycle until the drug is removed and the cleaved DNA is repaired. Inhibitors of these enzymes can affect many aspects of cell metabolism including replication, transcription, recombination, and chromosomal segregation.
D. Lactone Form Stabilizes HECPT
Utilizing HPLC and NMR techniques, researchers have demonstrated that camptothecin, CPT-11, and HECPT undergo an alkaline, pH-dependent hydrolysis of the E-ring lactone. The slow reaction kinetics allow one to assess whether both the lactone and non-lactone forms of the drug stabilizes the topoisomerase I-cleaved DNA complex. Studies indicate that only the closed lactone form of the drug helps stabilize the cleavable complex. This observation provides reasoning for the high degree of CPT-11 and HECPT activity observed in solid tumor models. Tumor cells, particularly hypoxic cells prevalent in solid neoplasms, have lower intracellular pH levels than normal cells. At pH levels below 7.0, the closed form of CPT-11 (and presumably of HECPT) predominates. Thus, the inventors predict that HECPT will be more effective at inhibiting topoisomerase I in an acidic environment than in cells having higher intracellular pH levels. It is the object of this invention to provide lactone stable HECPT as the basis of the claimed subject matter. Lactone stable HECPT is defined as HECPT which is dissolved in DMI or DMA in the presence of a pharmaceutically acceptable acid. The presence of the acid stabilizes the lactone form of HECPT. For the purpose of this invention lactone stable HECPT and HECPT are used interchangeably.
E. Camptothecin, CPT-11 and Topotecan
In 1966, Wall and Wani isolated camptothecin from the plant, Camptotheca acuminata. In the early 1970's, camptothecin reached Phase I trials and was found to have antitumor activity, but it caused unpredictable myelosuppression and hemorrhagic cystitis. Phase II studies with sodium camptothecin were limited because they induced unpredictable and severe myelosuppression, gastrointestinal toxicity, hemorrhagic cystitis, and alopecia. Clinical trials with sodium camptothecin were eventually discontinued because of unpredictable toxicities.
Two camptothecin derivatives, CPT-11 and Topotecan, have less sporadic toxicities but retain significant activity of the parent compound. CPT-11 and Topotecan are currently undergoing Phase I and Phase II development in the United States. 10, 11 methylene dioxycamptothecin is reportedly very active in preclinical studies, but it is also reported to be relatively insoluble in water which limits its use in the clinic (Pommier, et al. 1992).
Tables 1 and 2 present data summarizing Phase I and Phase II clinical trials of CPT-11. Neutropenia and diarrhea are the major reported, dose-limiting toxicities of CPT-11.
TABLE 1 __________________________________________________________________________ PHASE I STUDIES CPT-11 # Investigator Schedule Pts Dose Toxicity Tumor Type __________________________________________________________________________ Clavel et al 90 min. 37 115 mg/m.sup.2 /d Neutropenia* Breast (1 PR) QD .times. 3 Q21 (33-115) diarrhea, Mesothelioma days nausea and (1 PR) vomiting, alopecia Culine et al 90 min. 59 150 mg/m.sup.2 /wk Neutropenia* esophagus Q21 days (50-150) diarrhea* (1PR) cervix vomiting, (1PR) renal alopecia (1PR) overian fatigue (1PR) stomatitis Neutropenia* Negoro et al 30 min 17 100 mg/m.sup.2 Diarrhea*, N/V, NS CLC (2PRs) infusion (50-150) alopecia, weekly liver dysfunction Ohe et al 120 hr CI 36 40 mg/m.sup.2 /d Diarrhea* None Q3 wks (5-40) nausea and vomiting, thrombo- cytopenia, anemia, liver dysfunction Diarrhea* Rothenberg et al 90 mg QW .times. 4 32 180 mg/m.sup.2 /wk Neutropenia, Colon Ca (2 Q42 days (50-180) nausea, PRs) vomiting, alopecia Rowinsky et al 90 min 32 240 mg/m.sup.2 Neutropenia* Colon Ca (1PR) infusion (100-345) vomiting, Cervix Ca Q21 day diarrhea abd. (1 PR) pain, flushing __________________________________________________________________________ *Dose Limiting Toxicity
TABLE 2 __________________________________________________________________________ CPT-11 PHASE II TRIALS Tumor Reported Investigator Type Schedule # Pts. Response Rate Toxicities __________________________________________________________________________ Fukuoka et al Untreated 100 mg/m.sup.2 weekday 73 (23/72) PRs Neutropenia Non Small 31.9% diarrhea, nausea, Cell Lung vomiting, anorexia, Cancer alopecia Masudu et al Refractory or 100 mg/m.sup.2 weekly 16 (7/15) PRs Neutropenia, diarrhea Relapsed Small 47% pneumonitis Cell Lung Ca (12.5%) Negoro et al Small Cell Lung 100 mg/m.sup.2 /week 41 2 CRs and 7 PRs Neutropenia (38.6%) Cancer 33.3% N/V (61.5%) diarrhea (53.8%) alopecia (40.0%) Ohono et al Leukemia/ 200 mg Q3 No resp. 62 ** Neutropenia (91%) Lymphoma 40 mg/m.sup.2 Q0 .times. 5 34% PR Thrombocytopenia 20 mg/m.sup.2 bid .times. 7 25% PR Gastrointestinal (76%) Shimada et al Colon cancer 100 mg/m.sup.2 /week or 17 6/17(PR) Neutropenia (53%) 150 mg/m.sup.2 /Q 2 wks 46% N/V (35%) diarrhea (24%) Takeuchi et al Cervical cancer 100 mg/m.sup.2 weekly 69 SCR Neutropenia (89%) 150 mg/m.sup.2 weeks 8PR N/V (51%) RR of 23.6% Diarrhea (39.1%) Alopecia (38.1%) __________________________________________________________________________ **see text
F. HECPT is the Active Metabolite of CPT-11
Preclinical data, obtained by Barilero et al. on animals and more recently on humans, suggest that HECPT is the active metabolite of CPT-11 in vivo. Several different researchers administered CPT-11 and HECPT intravenously during Phase I trials and recorded the peak plasma concentrations (CpMax) at the end of the infusions. An analysis of the published mean peak plasma concentrations indicates that approximately 1.5% to 9% of the administered CPT-11 (on a per/mg basis) is converted into HECPT. The pharmacokinetic data from 30-minute intravenous infusions show a lower percentage of conversion (.about.1.5% ) of CPT-11 to HECPT than that observed following more prolonged infusions (.about.9% at 40 mg/m.sup.2 /d.times.5). The reported half life of HECPT observed in humans following the administration of CPT-11 ranges from 8.8 to 39.0 hours.
The biochemical and pharmacological relationship between CPT-11 and HECPT, as well as the role these compounds play in killing cancer cells in vivo is not completely understood. Investigators studying in vitro tumor cell lines have reported that HECPT has a 3600-fold greater inhibitory activity than CPT-11 against topoisomerase I in P388 cells and that HECPT is approximately 1000-fold more potent in generating single-strand DNA breaks in MOLT3 cells (Kawato, et al (1991)). However, Kaneda et al. report that HECPT has little anti-tumor activity compared to CPT-11 in vivo. They base their findings on studies conducted using an intermittent bolus schedule (days 1, 5, and 9) and an i.p. route of administration with an intraperitoneal P388 tumor model in mice.
Ohe et al. suggest that HECPT is a more toxic moiety of CPT-11 and could be responsible for much of the toxicity attributed to CPT-11. However, these same investigators noted a lack of correlation between HECPT pharmacokinetics and dose or CPT-11 pharmacokinetics and toxicity in human subjects. Furthermore, Ohe et al. noted a large range of interpatient variability in the AUC of CPT-11 and its metabolism to HECPT, which may result in unpredictable variability in the pharmacokinetic behavior, clinical anti-tumor effects, and toxicity in the individual patient. The data Ohe et al. obtained (using a 5-day, continuous intravenous infusion of CPT-11) also suggests that the conversion of CPT-11 to HECPT is a saturable process. If this is so, the clinical approach to maximizing dose intensity of the active metabolite would impose additional limitations on the effective use of CPT-11.
In preclinical studies of xenografts of human tumors in nude mice, Kawato et al. report that the sensitivity of human tumors to CPT-11 is independent of their ability to produce HECPT and that the effectiveness of CPT-11 is not related to the ability of the tumor to produce HECPT. Kawato et al. suggests that HECPT production is likely to be mediated in the plasma or interstitial compartment. Kaneda et al. observed that the plasma concentration of HECPT in mice was maintained longer after CPT-11 administration than after treatment with HECPT and suggested that clinicians should maintain plasma levels of HECPT to enhance the antitumor activity of CPT-11.
One of the advantages of present invention provides clinicians with the ability to directly adjust the plasma levels of HECPT to the point of therapeutic tolerance by controlling the dose and the schedule of administration. The inventors contend that this should lead to a superior ability to achieve better antitumor activity and reduce interpatient variability of the plasma levels of HECPT.
The different observations made in these studies suggest that direct administration of HECPT by parenteral and oral administration could provide significant clinical benefit for the treatment of cancer. However, in the past, HECPT has been considered insufficiently water soluble for clinical use. The current invention overcomes the solubility problem by providing lactone stable pharmaceutically acceptable multisolvent formulations of HECPT for parenteral use and also oral HECPT formulations.